My laboratory efforts have focused on the use of gene transfer for the treatment of cancer. Gene therapy for cancer includes strategies that replace defective tumor suppressor genes, transfer antisense molecules in tumors to inhibit expression transforming oncogenes, enhance the expression of immune co-stimulatory molecules and cytokines, or transfers genes into tumors encoding enzymes that locally activate non-toxic drugs into cytotoxic agents. Despite early optimism, few responses have been reported in clinical trials of a number of these approaches. A major reason for this is the low efficiency of in vivo gene transfer achieved by most vectors and the inability to target remote sites of tumor. My laboratory has focused on expanding cancer gene therapy from what is essentially a local therapeutic strategy to a systemic vaccination approach.As a primary effort in our laboratory, we investigated the use of genetically modified dendritic cells as an antitumor vaccination strategy. Most gene therapy of cancer represents a local treatment that is limited in its ability to target distant metastatic disease. The Metabolism Branch is involved in ongoing clinical trials studying the use of antitumor dendritic cell (DC) vaccines. In these studies, patients receive vaccinations with autologous dendritic cells that have been loaded ex vivo with peptide epitopes based on mutations found in the oncogenes or tumor suppressor genes expressed in the cancer in hopes of stimulating specific CTL responses against the tumor. This strategy is predicated upon knowing the patient's MHC-type and the affinity of the peptide vaccine sequence for a specific MHC molecule. In the laboratory we have been exploring an alternative approach involving the transfer and expression of tumor-associated antigen (TAA) genes into DC's. Vaccination with genetically altered DC's using recombinant adenoviruses has the potential advantages of: (1) expressing the TAA without the requirement for a detailed knowledge of a particular epitope or its binding affinity to a particular MHC molecule; (2) the ability to express larger antigenic sequences allows for the expression of a greater number of epitopes against which an immune response may be directed; (3) gene transfer and constitutive expression provides a continuous source of antigen to be loaded on to the MHC molecules overcoming the problem of low affinity epitopes that are strongly bound to MHC; (4) DC's may naturally process the expressed TAA leading to more "effective" epitopes, and (5) the viral proteins associated with the vector may stimulate DC maturation, activation, and recruitment of other immune cells by providing additional "danger signals" to the DC and the immune system.We have been working on adenoviral-mediated transfer of genes encoding tumor antigens into DC's that are then used to vaccinate our animal models against tumor. We studied this approach using the neu and K-ras oncogenes as targets. Dr. Yoshio Sakai, M.D., Ph.D., who recently left the laboratory, generated recombinant adenoviral vectors encoding the neu oncogene (homolog of human HER-2/neu) extracellular and transmembrane domains (ECDtm), extracellular only (ECD), green fluorescent protein (GFP), and a control vector expressing no transgene (Ad.null). Using Ad.GFP as a reporter, we found that murine bone marrow-derived DC's could be efficiently infected and the transgene expressed at relatively low concentrations of recombinant adenovirus. Infection of immature DC's induced the maturation and co-culture with transduced DC's stimulated greater proliferation of nave splenocytes in mixed lymphocyte culture compared to uninfected DC's or those infected with the Ad.null control vector indicating increased DC activation. We then studied antitumor vaccination using dendritic cells transduced with these vectors in a transgenic mouse model of neu induced breast cancer. BALB-neuT mice transgenic for the rat neu oncogene spontaneously develop breast cancers beginning at 14 to 15 weeks of age and continue to develop tumors until 100% of the mice and 100% of the mammary glands have developed tumors by 25-weeks of age. We found that the longer antigenic construct, ECDtm, offered more protection from tumor compared to the shorter ECM. Using this approach, over 67% of the mice remained tumor free at 28-weeks of age compared to none of the mice in the groups that were treated with DC's alone or with DC's transduced with Ad.null. In addition, even in those mice that developed tumors, the average number of tumors per mouse was significantly reduced. Vaccination induced anti-neu antibodies that appear to be protective against tumor. Depletion studies indicated that CD4+, but not CD8+ T cells are critical for the vaccine's effectiveness. The protective effect of the Ad.Neu-DC vaccination was not affected by pre-existing immunity to adenovirus, but the efficacy of a direct vaccination using the Ad.Neu vector alone was significantly reduced. This work was the subject of talks presented at the American Society for Gene Therapy and the Presidential Session of the International Society for Biological Therapy, both in 2003 and is currently in press in Cancer Research. In collaboration with Jong-Myun Park, Ph.D. in the laboratory of Dr. Jay Berzofsky, M.D., Ph.D. we examined the mechanism of protection using the direct Ad.Neu vaccine approach. Direct vaccination with this vector is also protective in the nave BALB-neuT mouse. This protection is lost in B cell deficient mice and the induction of the IgG2a immunoglobulin subtype appeared the most effective in preventing tumors. These studies confirmed an important early role for CD4+ T cells and antibody rather than C8+ CTL in generating anti-neu immunity in this model. The second study has been submitted as manuscript for publication. The effectiveness of either vaccine approach is attenuated as the BALB-neuT mice age, or if tumors have already developed. We plan further studies looking at ways to block the activity of CD4+ T regulatory cells that may function to inhibit the immune response in older animals and those with a tumor burden. In addition, we are discussing the possibility of developing a clinical trial of a recombinant adenovirus expressing a non-signaling human HER-2/neu-DC vaccine trial in patients with HER-2/neu expressing breast cancers. In an effort to test the broader application of this approach we intend to explore this vaccine approach in other oncogene transgenic tumor models most notably K-ras.In another series of experiments Dr. Sakai and Brian Morrison, a student in the lab generated an adenovirus expressing murine interleukin-12 (Ad.mIL-12). DC's infected with relatively small amounts of Ad.mIL-12 secreted significant levels of mIL-12 and was able to inhibit the development of tumors when mice were challenged injections of MC38 mouse colon carcinoma cells. When co-cultured with nave splenocytes the genetically altered DC induced interferon-gamma secretion at levels that could not be achieved even with the addition of up to 300 ng/mL of recombinant IL-12 protein suggesting that this is a potent combination that may skew the immune response toward a CTL response (Th1). Studies are in progress to determine if the protection from tumor is attributable solely to the IL-12 secretion by these cells, or if it is necessary to have DC's and secretion of IL-12 to generate the protection.In the clinical arena, through a CRADA collaboration with NewLink Genetics of Ames, IA, we developed a clinical vaccine trial using allogeneic lung cancer cells that have been genetically altered with a Moloney murine retrovirus vector to express alpha (1,3) galactosyl transferase (alpha-GT) for patients with advanced non-small cell lung cancer.